Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB) infects 1.7 billion people worldwide. Three million people die of TB annually. This makes TB the leading cause of death associated with infectious diseases globally. Factors involved in the regulation of many phenotypes of MTB such as virulence, latency, antibiotic resistance and ability to survive under harsh conditions are not well understood. Bacterial cell density dependent signaling (quorum sensing) has been shown to play a major role in the control of virulence, sporulation, cell division, biofilm development and drug resistance in bacteria. Data on the role of quorum sensing in the regulation of MTB complex phenotypes are limited. Two recent studies, however, have demonstrated a conserved mechanism of extracellular signaling in eukaryotes and prokaryotes. This signaling system consists of an intramembrane signal precursor that is cleaved by an intramembrane protease to release an active extracellular peptide signal. In Drosophila, Rhomboid is the intramembrane serine protease that catalyses the release of epidermal growth factor receptor (EGFr) ligands. The EGFr signals are required for proper cell differentiation, growth and development in animals. In Drosophila, the EGFr signaling has been shown to be crucial for the correct development of the compound eye and wing veination. In Providencia stuartii, a bacterium, a Rhomboid-like protein, AarA, is required for production of an extracellular peptide-like signal. Two Rhomboid-like sequences have been demonstrated in the MTB genome and M. smegmatis. To gain insight into the role of cell-to-cell signaling in MTB, the role of the Rhomboid-like proteins in mycobacteria will be investigated using M. smegmatis as a model. The specific objectives include: (1) To investigate the components of the Rhomboid signaling pathway in mycobacteria. This will be accomplished by determining whether the Rhomboid homologues in mycobacteria can substitute the Rhomboid homologue (AarA) in P. stuartii. (2) To use genetic techniques to examine the role of the Rhomboid-like proteins in the physiology of mycobacteria. This will be addressed by construction of deletion mutation in M. smegmatis Rhomboid homologues and determining how this affects signal production, morphology, survival and other phenotypes of mycobacteria. (3) To identify quorum sensing regulated gene fusions in mycobacteria. This will be achieved through construction of random lacZ reporter transcriptional gene fusions in M. smegmatis chromosome. Fusions whose activity changes at high cell density will be identified and mapped as those regulated by quorum sensing. This study will provide information on mycobacterial functions that are regulated by cell-to-cell communication. This will expand our understanding of mycobacterial biology and may provide novel candidate vaccines and drug targets.